Dual amylin and calcitonin receptor agonists and uses thereof

ABSTRACT

The present disclosure related to the field of medicine. More particularly, the disclosure is in the field of treatment of diabetes, obesity, and/or dyslipidemia. The disclosure relates to compounds that agonize both the calcitonin and amylin receptors and can lower food intake, body weight, glucose and/or triglycerides, so can be used to treat diabetes, obesity and/or dyslipidemia. The present disclosure also includes pharmaceutical compositions containing such compounds and therapeutic uses of such compounds and compositions.

The present disclosure relates to the field of medicine. Moreparticularly, the disclosure is in the field of treatment of diabetes,obesity, non-alcoholic steatohepatitis (NASH), and/or dyslipidemia. Thedisclosure relates to compounds that agonize both the amylin andcalcitonin receptors and can therefore lower food intake, body weight,glucose, and/or triglycerides, and may be used to treat diabetes,obesity, NASH, and/or dyslipidemia. The present disclosure also includespharmaceutical compositions containing such compounds, and therapeuticuses of such compounds and pharmaceutical compositions.

Amylin is a peptide hormone that is co-secreted with insulin from thepancreatic β-cells and is deficient in people with diabetes. It inhibitsglucagon secretion, delays gastric emptying, and acts as a satietyagent. An amylin analog, pramlintide, is available to treat diabeticpatients who use insulin because it lowers blood sugar levels. Theelimination half-life of this medicine is less than an hour and it isused at mealtimes, so a patient needs multiple doses in one day to usethis medicine in therapy.

Calcitonin is a hormone that is produced in the thyroid gland that playsa role in regulating levels of calcium and phosphate in the blood.Salmon calcitonin is available to treat conditions with high levels ofcalcium in the blood, such as hypercalcemia. Salmon calcitonin has ashort half-life of less than two hours, so a patient needs to dose oncea day or multiple times a day to use this peptide in therapy.

It has been shown that compounds which agonize both the amylin andcalcitonin receptors have positive effects such as lowering bloodglucose levels and inducing weight loss. See WO2016/034604,WO2015/071229 and WO2010/085700. The natural half-life of known amylinand calcitonin receptor agonists is short, so an extended time action isdesirable. However, chemical modifications intended to extend activitytime have also, thus far, shown a decrease in potency. In addition,agonists of the amylin and calcitonin receptors have stabilitychallenges due to their tendency to fibrillate and the presence of alabile disulfide bond at neutral pH.

There is a need for alternative compounds that agonize both the amylinand calcitonin receptors. In addition, the need exists for dual amylinand calcitonin receptor agonists with extended time action and preservedpotency. Therapeutically desirable compounds would agonize both theamylin and calcitonin receptors and provide one or more advantageousproperties such as lowering food intake, lowering body weight, loweringblood glucose levels, lowering triglycerides, and/or lowering insulinlevels. In addition, therapeutically desirable compounds may have one ormore additional advantageous properties such as extended time actionwith preserved or improved potency in agonizing both the amylin andcalcitonin receptors, a low risk of immunogenic response, chemicalstability at a neutral pH, and/or a low risk of fibrillation.

Further, combination of a dual amylin and calcitonin receptor agonist ofthe present disclosure, optionally combined with an incretin or incretinanalog, is desired to provide treatment for diabetes, obesity, NASH,and/or dyslipidemia. Such combination will also preferably be moreeffective than either molecule alone. For example, such treatment withsuch combination may allow for use of lower doses of either or bothmolecules as compared to each molecule alone, potentially leading tolower side effects (or a shorter duration of one or the other therapy)while maintaining efficacy. It is believed that the novel combination(s)provided herein will be effective treatments for diabetes, obesity,NASH, and/or dyslipidemia.

Accordingly, the present disclosure provides a method of treatingdiabetes, obesity, NASH, and/or dyslipidemia, comprising administeringto a patient in need of such treatment an effective amount of a dualamylin and calcitonin receptor agonist with an effective amount of anincretin or incretin analog. The present disclosure further provides amethod of treating clinical or pre-clinical diabetes, obesity, NASH,and/or dyslipidemia comprising administering to a patient in need ofsuch treatment an effective amount of a dual amylin and calcitoninreceptor agonist in combination with an effective amount of incretin orincretin analog.

Certain presently disclosed compounds which agonize the amylin andcalcitonin receptors show an effective reduction in food intake and bodyweight, as well as glucose and insulin lowering. Further, certainpresently disclosed compounds show a decreased risk of immunogenicityand a low level of fibrillation. The pharmacokinetic properties ofcertain presently disclosed compounds show greatly extended half-life,which will enable dosing of once a week of these compounds for use intherapy. Other embodiments of the present disclosure are useful formaking such compounds for use in therapy. Methods for combining thepresently disclosed compounds with incretins or incretin analogs arealso disclosed.

One of the compounds of the disclosure, Compound I, comprises thefollowing sequence: Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH₂ (SEQID NO:1), or a pharmaceutically acceptable salt thereof. Below is adepiction of the structure of Compound I using the standard singleletter amino acid codes with the exception of residues Acetyl-A1, Aib14,and P32-NH₂, where the structures of these amino acid residues have beenexpanded:

Accordingly, one of the compounds of the present disclosure is:

or a pharmaceutically acceptable salt thereof.

The compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, may further comprise one of more additional elements toextend the half-life of the compound. Elements that could be added to acompound comprising SEQ ID NO:1 for this purpose, with or without alinker and at any suitable position in the sequence, include the Fcportion of an immunoglobulin, fragments of the Fc portion of animmunoglobulin, human serum albumin (HSA), VHH, a variant of a VHH(variable domain of heavy chain antibodies) nanobody, fragments of humanserum albumin, a C₁₆-C₂₀ monoacid, a C₁₆-C₂₀ diacid, and a polyethyleneglycol (PEG) moiety or PEG alternative (e.g., polysarcosine). Otherelements that could be added to a compound comprising SEQ ID NO:1 forthis purpose, with or without a linker and at any suitable position inthe sequence can also include, a C₁₆-C₂₂ monoacid, or a C₁₆-C₂₂ diacid,a C₁₈-C₂₀ monoacid, or a C₁₈-C₂₀ diacid.

In one embodiment of the presently disclosed compounds is a compoundcomprising SEQ ID NO:1 wherein the compound further comprises a fattyacid moiety with or without a linker.

In another embodiment, the presently disclosed compounds have an acetylgroup (CH₃CO) on the alanine amino acid residue at the N-terminus of thepeptide. This is signified herein by the phrase “Acetyl” and a dashpreceding the N terminal amino acid residue in the peptide description.The presently disclosed compounds also have an amide group (NH₂) on theC terminal amino acid residue, proline. This is signified herein by theformula “NH₂” and a dash after the C terminal amino acid residue in thepeptide description.

In another embodiment, a compound comprising SEQ ID NO:1 is acylated atany suitable amino acid in the sequence and consequently has an extendedhalf-life. In another embodiment, a compound comprising SEQ ID NO:1 isacylated at the epsilon amino group on the lysine sidechain andconsequently has an extended half-life. In a specific embodiment, acompound comprising SEQ ID NO:1 is acylated at the epsilon amino groupon the lysine sidechain according to the formula2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H.Accordingly, one of the compounds of the present disclosure is CompoundII, comprising the following sequenceAcetyl-ASHLSTAVLGK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LS-Aib-ELHKLEDYPRTDVGAESP-NH₂(SEQ ID NO:2), or a pharmaceutically acceptable salt thereof.

In a specific embodiment, a compound consisting of SEQ ID NO:1 isacylated at the epsilon amino group on the lysine sidechain according tothe formula2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H.Accordingly, one of the compounds of the present disclosure is CompoundII, consisting of the following sequence

Acetyl-ASHLSTAVLGK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LS-Aib-ELHKLEDYPRTDVGAESP-NH₂(SEQ ID NO:2), or a pharmaceutically acceptable salt thereof. In aspecific embodiment, a compound consisting essentially of SEQ ID NO:1 isacylated at the epsilon amino group on the lysine sidechain according tothe formula2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H.Accordingly, one of the compounds of the present disclosure is CompoundII, consisting essentially of the following sequenceAcetyl-ASHLSTAVLGK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LS-Aib-ELHKLEDYPRTDVGAESP-NH₂(SEQ ID NO:2), or a pharmaceutically acceptable salt thereof. Below is adepiction of the structure of Compound II using the standard singleletter amino acid codes with the exception of residues Acetyl-A1, K11,Aib14, and P32-NH₂, where the structures of these amino acid residueshave been expanded.

Accordingly, one of the compounds of the present disclosure is:

or a pharmaceutically acceptable salt thereof.

Another embodiment of the present disclosure provides a pharmaceuticalcomposition comprising the compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients. Another embodiment of thepresent disclosure provides a pharmaceutical composition comprising thecompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

Another embodiment of the present disclosure provides a method oftreating diabetes in a patient in need thereof, comprising administeringto the patient an effective amount of a compound comprising SEQ ID NO:1,or a pharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure provides a method of treating diabetes in a patientin need thereof, comprising administering to the patient an effectiveamount of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof. A preferred embodiment of the presentdisclosure provides a method of treating Type 2 diabetes in a patient inneed thereof, comprising administering to the patient an effectiveamount of a compound comprising SEQ ID NO:1, or a pharmaceuticallyacceptable salt thereof. Another preferred embodiment of the presentdisclosure provides a method of treating Type 2 diabetes in a patient inneed thereof, comprising administering to the patient an effectiveamount of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof. Another embodiment of the present disclosureprovides a method of treating diabetes in a patient in need thereof,comprising administering to the patient a pharmaceutical compositioncomprising a compound comprising SEQ ID NO:1. Another embodiment of thepresent disclosure provides a method of treating diabetes in a patientin need thereof, comprising administering to the patient apharmaceutical composition comprising a compound comprising SEQ ID NO:2.A preferred embodiment of the present disclosure provides a method oftreating Type 2 diabetes in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another preferred embodiment of thepresent disclosure provides a method of treating Type 2 diabetes in apatient in need thereof, comprising administering to the patient apharmaceutical composition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oftreating obesity in a patient in need thereof, comprising administeringto the patient an effective amount of a compound comprising SEQ ID NO:1,or a pharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure provides a method of treating obesity in a patient inneed thereof, comprising administering to the patient an effectiveamount of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof. Another embodiment of the present disclosureprovides a method of treating obesity in a patient in need thereof,comprising administering to the patient a pharmaceutical compositioncomprising a compound comprising SEQ ID NO:1. Another embodiment of thepresent disclosure provides a method of treating obesity in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oftreating dyslipidemia in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oftreating dyslipidemia in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oftreating dyslipidemia in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another embodiment of the presentdisclosure provides a method of treating dyslipidemia in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oftreating NASH in a patient in need thereof, comprising administering tothe patient an effective amount of a compound comprising SEQ ID NO:1, ora pharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure provides a method of treating NASH in a patient inneed thereof, comprising administering to the patient an effectiveamount of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof. Another embodiment of the present disclosureprovides a method of treating NASH in a patient in need thereof,comprising administering to the patient a pharmaceutical compositioncomprising a compound comprising SEQ ID NO:1. Another embodiment of thepresent disclosure provides a method of treating NASH in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2

Another embodiment of the present disclosure provides a method oflowering food intake in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering food intake in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering food intake in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another embodiment of the presentdisclosure provides a method of lowering food intake in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oflowering body weight in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering body weight in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering body weight in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another embodiment of the presentdisclosure provides a method of lowering body weight in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oflowering blood glucose in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering blood glucose in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID:2, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering blood glucose in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another embodiment of the presentdisclosure provides a method of lowering blood glucose in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oflowering triglycerides in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering triglycerides in a patient in need thereof, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.Another embodiment of the present disclosure provides a method oflowering triglycerides in a patient in need thereof, comprisingadministering to the patient a pharmaceutical composition comprising acompound comprising SEQ ID NO:1. Another embodiment of the presentdisclosure provides a method of lowering triglycerides in a patient inneed thereof, comprising administering to a patient a pharmaceuticalcomposition comprising a compound comprising SEQ ID NO:2.

Another embodiment of the present disclosure provides a method oftreating diabetes in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of treating diabetes in apatient in need thereof, wherein the compound comprising SEQ ID NO:2 isadministered once weekly.

Another embodiment of the present disclosure provides a method oftreating Type 2 diabetes in a patient in need thereof, wherein thecompound comprising SEQ ID NO:1 is administered once weekly. Anotherembodiment of the present disclosure provides a method of treating Type2 diabetes in a patient in need thereof, wherein the compound comprisingSEQ ID NO:2 is administered once weekly.

Another embodiment of the present disclosure provides a method oftreating obesity in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of treating obesity in apatient in need thereof, wherein the compound comprising SEQ ID NO:2 isadministered once weekly.

Another embodiment of the present disclosure provides a method oftreating dyslipidemia in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of treating dyslipidemia ina patient in need thereof, wherein the compound comprising SEQ ID NO:2is administered once weekly.

Another embodiment of the present disclosure provides a method oftreating NASH in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of treating NASH in apatient in need thereof, wherein the compound comprising SEQ ID NO:2 isadministered once weekly.

Another embodiment of the present disclosure provides a method oflowering food intake in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of lowering food intake in apatient in need thereof, wherein the compound comprising SEQ ID NO:2 isadministered once weekly.

Another embodiment of the present disclosure provides a method oflowering body weight in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:1 is administered once weekly. Another embodimentof the present disclosure provides a method of lowering body weight in apatient in need thereof, wherein the compound comprising SEQ ID NO:2 isadministered once weekly.

Another embodiment of the present disclosure provides a method oflowering blood glucose in a patient in need thereof, wherein thecompound comprising SEQ ID NO:1 is administered once weekly. Anotherembodiment of the present disclosure provides a method of lowering bloodglucose in a patient in need thereof, wherein the compound comprisingSEQ ID NO:2 is administered once weekly.

Another embodiment of the present disclosure provides a method oflowering triglycerides in a patient in need thereof, wherein thecompound comprising SEQ ID NO:1 is administered once weekly. Anotherembodiment of the present disclosure provides a method of loweringtriglycerides in a patient in need thereof, wherein the compoundcomprising SEQ ID NO:2 is administered once weekly.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in therapy. Another embodiment of the presentdisclosure provides a pharmaceutical composition comprising SEQ ID NO:1for use in therapy. Another embodiment of the presently disclosedcompounds provides a compound comprising SEQ ID NO:2, or apharmaceutically acceptable salt thereof, for use in therapy. Anotherembodiment of the present disclosure provides a pharmaceuticalcomposition comprising SEQ ID NO:2 for use in therapy.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in the treatment of diabetes. Another embodiment of thepresent disclosure provides a pharmaceutical composition comprising SEQID NO:1 for use in the treatment of diabetes. Another embodiment of thepresently disclosed compounds provides a compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, for use in thetreatment of diabetes. Another embodiment of the present disclosureprovides a pharmaceutical composition comprising SEQ ID NO:2 for use inthe treatment of diabetes.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in the treatment of Type 2 diabetes. Another embodimentof the present disclosure provides a pharmaceutical compositioncomprising SEQ ID NO:1 for use in the treatment of Type 2 diabetes.Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof, for use in the treatment of Type 2 diabetes. Another embodimentof the present disclosure provides a pharmaceutical compositioncomprising SEQ ID NO:2 for use in the treatment of Type 2 diabetes.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in the treatment of obesity. Another embodiment of thepresent disclosure provides a pharmaceutical composition comprising SEQID NO:1 for use in the treatment of obesity. Another embodiment of thepresently disclosed compounds provides a compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, for use in thetreatment of obesity. Another embodiment of the present disclosureprovides a pharmaceutical composition comprising SEQ ID NO:2 for use inthe treatment of obesity.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in the treatment of dyslipidemia. Another embodiment ofthe present disclosure provides a pharmaceutical composition comprisingSEQ ID NO:1 for use in the treatment of dyslipidemia. Another embodimentof the presently disclosed compounds provides a compound comprising SEQID NO:2, or a pharmaceutically acceptable salt thereof, for use in thetreatment of dyslipidemia. Another embodiment of the present disclosureprovides a pharmaceutical composition comprising SEQ ID NO: 2, for usein the treatment of dyslipidemia.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, for use in the treatment of NASH. Another embodiment of thepresent disclosure provides a pharmaceutical composition comprising SEQID NO:1 for use in the treatment of NASH.

Another embodiment of the presently disclosed compounds provides acompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof, for use in the treatment of NASH. Another embodiment of thepresent disclosure provides a pharmaceutical composition comprising SEQID NO:2 for use in the treatment of NASH.

Another embodiment of the present disclosure provides for the use of acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment ofdiabetes. Another embodiment of the present disclosure provides for theuse of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament for thetreatment of diabetes. A preferred embodiment of the present disclosurecompounds provides for the use of a compound comprising SEQ ID NO:1, ora pharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of Type 2 diabetes. In another preferredembodiment of the present disclosure provides for the use of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for the treatment of diabetes.

Another embodiment of the present disclosure provides for the use of acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment ofobesity. Another embodiment of the present disclosure provides for theuse of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament for thetreatment of obesity.

Another embodiment of the present disclosure provides for the use of acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment ofdyslipidemia. Another embodiment of the present disclosure provides forthe use of a compound comprising SEQ ID NO:2, or a pharmaceuticallyacceptable salt thereof, in the manufacture of a medicament for thetreatment of dyslipidemia.

Another embodiment of the present disclosure provides for the use of acompound comprising SEQ ID NO:1, or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment of NASH.Another embodiment of the present disclosure provides for the use of acompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for the treatment of NASH.

Another embodiment of the presently disclosed compounds is the compoundof SEQ ID NO:1 where the half-life (tv_(1/2)) is extended.

Another embodiment of the present disclosure is a method of treating acondition in patient in need thereof, selected from the group consistingof: diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to the patient an effective amount of a compoundcomprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof,in combination with an effective amount of an incretin or incretinanalog. Another embodiment of the present disclosure is a method oftreating a condition in a patient in need thereof, selected from thegroup consisting of: diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to the patient an effective amount of acompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof, in combination with an effective amount of an incretin orincretin analog.

In a particular embodiment of the present disclosure is a method oftreating a condition in patient in need thereof, selected from a groupconsisting of: diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with a GLP-1 agonist. In a particularembodiment of the present disclosure is a method of treating a patientwith diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with a GLP-1 agonist selected from thegroup consisting of compounds comprising Compound III (SEQ ID NO:6),Compound VIII (SEQ ID NO:12), or Compound IX (SEQ ID NO:13). In aparticular embodiment of the present disclosure is a method of treatinga patient with diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:2, or a pharmaceutically acceptablesalt thereof, in combination with a GLP-1 agonist. In a particularembodiment of the present disclosure is a method of treating a patientwith diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:2, or a pharmaceutically acceptablesalt thereof, in combination with a GLP-1 agonist selected from thegroup consisting of compounds comprising Compound III (SEQ ID NO:6),Compound VIII (SEQ ID NO:12), or Compound IX (SEQ ID NO:13).

In a particular embodiment of the present disclosure is a method oftreating a patient with diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to a patient in need of such treatment aneffective amount of a compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, in combination with a dualGIP/GLP-1 agonist. In a particular embodiment of the present disclosureis a method of treating a patient with diabetes, obesity, NASH, and/ordyslipidemia, comprising administering to a patient in need of suchtreatment an effective amount of a compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, in combination with a dualGIP/GLP-1 agonist selected from the group consisting compoundscomprising Compound VI (SEQ ID NO:9) and Compound VII (SEQ ID NO:10). Ina particular embodiment of the present disclosure is a method oftreating a patient with diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to a patient in need of such treatment aneffective amount of a compound comprising SEQ ID NO:2 in combinationwith a dual GIP/GLP-1 agonist. In a particular embodiment of the presentdisclosure is a method of treating a patient with diabetes, obesity,NASH, and/or dyslipidemia, comprising administering to a patient in needof such treatment an effective amount of a compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, in combination witha dual GIP/GLP-1 agonist selected from the group consisting of compoundscomprising Compound VI (SEQ ID NO:9) and Compound VII (SEQ ID NO:10).

In a particular embodiment of the present disclosure is a method oftreating a patient with diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to a patient in need of such treatment aneffective amount of a compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, in combination with atriagonist of glucagon, GIP, and GLP-1. In a particular embodiment ofthe present disclosure is a method of treating a patient with diabetes,obesity, NASH, and/or dyslipidemia, comprising administering to apatient in need of such treatment an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof,in combination with a triagonist of glucagon, GIP, and GLP-1. In aparticular embodiment of the present disclosure is a method of treatinga patient with diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with a triagonist of glucagon, GIP, andGLP-1 comprising Compound V (SEQ ID NO:8). In a particular embodiment ofthe present disclosure is a method of treating a patient with diabetes,obesity, NASH, and/or dyslipidemia, comprising administering to apatient in need of such treatment an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof,in combination with a triagonist of glucagon, GIP, and GLP-1 comprisingCompound V (SEQ ID NO:8).

In a particular embodiment of the present disclosure is a method oftreating a patient with diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to a patient in need of such treatment aneffective amount of a compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, in combination with an analogof oxyntomodulin. In a particular embodiment of the presently disclosedcompounds is a method of treating a patient with diabetes, obesity,NASH, and/or dyslipidemia, comprising administering to a patient in needof such treatment an effective amount of a compound comprising SEQ IDNO:2 in combination with an analog of oxyntomodulin. In a particularembodiment of the present disclosure is a method of treating a patientwith diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with an analog of oxyntomodulin comprisingCompound IV (SEQ ID NO:7). In a particular embodiment of the presentdisclosure is a method of treating a patient with diabetes, obesity,NASH, and/or dyslipidemia, comprising administering to a patient in needof such treatment an effective amount of a compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, in combination withan analog of oxyntomodulin comprising Compound IV (SEQ ID NO:7).

Another embodiment of the present disclosure is a method of treating apatient with diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with an incretin or incretin analog whereinthe compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, and the incretin or incretin analog are administeredsimultaneously. Another embodiment of the present disclosure is a methodof treating a patient with diabetes, obesity, NASH, and/or dyslipidemia,comprising administering to a patient in need of such treatment aneffective amount of a compound comprising SEQ ID NO:2, or apharmaceutically acceptable salt thereof, in combination with anincretin or incretin analog wherein the compound comprising SEQ ID NO:2,or a pharmaceutically acceptable salt thereof, and the incretin orincretin analog are administered simultaneously.

Another embodiment of the present disclosure is the method of treating apatient with diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with an incretin or incretin analog whereinthe compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, is administered prior to the administration of theincretin or incretin analog. Another embodiment of the presentdisclosure is the method of treating a patient with diabetes, obesity,NASH, and/or dyslipidemia, comprising administering to a patient in needof such treatment an effective amount of a compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, in combination withan incretin or incretin analog wherein the compound comprising SEQ IDNO:2, or a pharmaceutically acceptable salt thereof, is administeredprior to the administration of the incretin or incretin analog.

Another embodiment of the present disclosure is the method of treating apatient with diabetes, obesity, NASH, and/or dyslipidemia, comprisingadministering to a patient in need of such treatment an effective amountof a compound comprising SEQ ID NO:1, or a pharmaceutically acceptablesalt thereof, in combination with an incretin or incretin analog whereinthe incretin or incretin analog is administered prior to theadministration of the compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure is the method of treating a patient with diabetes,obesity, NASH, and/or dyslipidemia, comprising administering to apatient in need of such treatment an effective amount of a compoundcomprising SEQ ID NO:2, or a pharmaceutically acceptable salt thereof,in combination with an incretin or incretin analog wherein the incretinor incretin analog is administered prior to the administration of thecompound comprising SEQ ID NO:2, or a pharmaceutically acceptable saltthereof.

Another embodiment of the present disclosure is the compound comprisingSEQ ID NO:1 for use in separate, simultaneous, or sequential combinationwith an incretin or incretin analog for the treatment of diabetes,obesity, NASH, and/or dyslipidemia. Another embodiment of the presentdisclosure is the compound comprising SEQ ID NO:2 for use in separate,simultaneous, or sequential combination with an incretin or incretinanalog for the treatment of diabetes, obesity, NASH, and/ordyslipidemia.

Another embodiment of the present disclosure provides for the use of thecompound comprising SEQ ID NO:1 in the manufacture of a medicament forthe treatment of diabetes. Another embodiment of the present disclosureprovides for the use of the compound comprising SEQ ID NO:2 in themanufacture of a medicament for the treatment of diabetes.

Another embodiment of the present disclosure provides for the use of thecompound comprising SEQ ID NO:1 in the manufacture of a medicament forthe treatment of Type 2 diabetes. Another embodiment of the presentdisclosure provides for the use of the compound comprising SEQ ID NO:2in the manufacture of a medicament for the treatment of Type 2 diabetes.

Another embodiment of the present disclosure provides for the use of thecompound comprising SEQ ID NO:1 in the manufacture of a medicament forthe treatment of obesity. Another embodiment of the present disclosureprovides for the use of the compound comprising SEQ ID NO:2 in themanufacture of a medicament for the treatment of obesity.

Another embodiment of the present disclosure provides for the use of thecompound comprising SEQ ID NO:1 in the manufacture of a medicament forthe treatment of dyslipidemia. Another embodiment of the presentdisclosure provides for the use of the compound comprising SEQ ID NO:2in the manufacture of a medicament for the treatment of dyslipidemia.

Another embodiment of the present disclosure provides for the use of thecompound comprising SEQ ID NO:1 in the manufacture of a medicament forthe treatment of NASH. Another embodiment of the present disclosureprovides for the use of the compound comprising SEQ ID NO:2 in themanufacture of a medicament for the treatment of diabetes.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the disclosure pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of dual amylin and calcitonin receptor agonists,pharmaceutical compositions, pharmaceutical combinations, the preferredmethods and materials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually means “at leastone.”

The term “analog” refers to a compound, such as a synthetic peptide orpolypeptide, that activates a target receptor and that elicits at leastone in vivo or in vitro effect elicited by a native agonist for thatreceptor.

The term “combination” as used herein means compounds presentlydisclosed may be used in simultaneous, separate or sequentialcombination with one or more additional therapeutic agents useful forinducing weight loss, treating diabetes, conditions related to diabetes,obesity, NASH, and/or dyslipidemia. Non-limiting examples of theadditional therapeutic agents that can be combined with the claimedcompounds include: insulin or insulin analogs; incretin compounds orincretin analogs, such as glucagon-like-peptide-1 (GLP-1) or GLP-1analogs, gastric inhibitory polypeptide (GIP) or GIP analogs,oxyntomodulin or oxyntomodulin analogs; dual GIP/GLP-1 agonists;Gcg/GIP/GLP-1 triagonists (triagonists of glucagon, GIP, and GLP-1); orcombinations of any of the foregoing agents. The claimed compounds andthe additional therapeutic agent(s) can be co-administered through thesame delivery route and device such as a single pill, capsule, tablet,or injectable formulation; or separately administered either at the sametime in separate delivery devices or routes; or administeredsequentially.

The term “diabetes” refers to a disease in which the body's ability toproduce or respond to the hormone insulin is impaired, resulting inabnormal metabolism of carbohydrates and elevated levels of glucose inthe blood and urine. As used herein, the term diabetes may refer to achronic condition that affects the way the body processes blood sugar,or glucose, e.g., type 2 diabetes mellitus (T2DM); a chronic conditionin which the pancreas produces little or no insulin, e.g., type 1diabetes mellitus (T1DM); a condition in which blood sugar is high, butnot high enough to be type 2 diabetes, e.g., pre-diabetes; a form ofhigh blood sugar affecting pregnant women, e.g., gestational diabetes.

The term “dyslipidemia” as used herein refers to a disorder oflipoprotein metabolism, including lipoprotein overproduction ordeficiency. Dyslipidemias may be manifested by elevation of the totalcholesterol, low-density lipoprotein (LDL) cholesterol and triglycerideconcentrations, and/or a decrease in the high-density lipoprotein (HDL)concentration in the blood. Dyslipidemia may or may not develop inconnection with diabetes.

The term “incretin” as used herein refers to a group of endogenousmetabolic hormones that are excreted from the enteroendocrine cells inthe stomach and pancreas to stimulate a decrease in blood glucose,typically via regulation of the amount of insulin that is secreted aftermeals. The term “incretin analog” as used herein refers to a group ofsynthetic incretin mimetics which are physiologically similar toincretins. Further, “incretin analogs” may have favorablepharmacological properties compared to endogenous incretins.

The term “treatment” or “treating” as used herein refers to themanagement and care of a patient having a condition for which amylin andcalcitonin receptor peptide agonist administration is indicated for thepurpose of combating or alleviating symptoms and complications of thecondition. Treating includes administering a compound or apharmaceutical composition containing a compound of the presentlydisclosed compounds to a patient in need thereof to prevent the onset ofsymptoms or complications, alleviating the symptoms or complications, oreliminating the disease, condition, or disorder. Preferably treatingincludes administering a compound or pharmaceutical compositioncontaining a compound of the presently disclosed compounds to a patientin need thereof to result in a net loss of body weight, a reduction infood intake, a reduction in blood glucose levels and/or a reduction intriglyceride levels. The patient to be treated is an animal, andpreferably a human being.

As used herein, the term “effective amount” refers to the amount or doseof a compound of the presently disclosed compounds or a pharmaceuticalcomposition containing the presently disclosed compounds, upon whichsingle or multiple dose administration to the patient will elicit thebiological or medical response or desired therapeutic effect beingsought by a health care professional. Preferably an effective amount ofa compound or pharmaceutical composition containing a compound of thepresently disclosed compounds administered to a patient in need thereofwould result in a net loss of body weight, a reduction in food intake, areduction in blood glucose levels and/or a reduction in triglyceridelevels. A dose can include a higher initial loading dose, followed by alower dose.

The term “patient,” refers to an animal, and preferably to a human. Incertain embodiments, the patient, preferably a human, is furthercharacterized with a disease, disorder or condition that would benefitfrom administration of a compound that agonizes both the amylin andcalcitonin receptors. Pharmaceutical compositions comprising thepresently disclosed compounds may be administered orally or parenterallyto patients in need of such treatment. Parenteral administration may beperformed by subcutaneous, intramuscular or intravenous injection bymeans of a syringe, optionally a pen-like syringe, or mechanical driveninjector. Alternatively, parenteral administration can be performed bymeans of an infusion pump. Embodiments of the presently disclosedcompounds provide pharmaceutical compositions suitable foradministration to a patient comprising administering to a patient inneed thereof a therapeutically effective amount of a compound presentlydisclosed and one or more pharmaceutically acceptable excipients. Suchpharmaceutical compositions may be prepared by any of a variety oftechniques using conventional excipients for pharmaceutical productswhich are well known in the art. (Remington's Pharmaceutical Sciences,21st Edition, University of the Sciences in Philadelphia, Philadelphia,Pa., USA (2006)).

The term “NASH” as used herein stands for non-alcoholic steatohepatitis,aka fatty liver disease. “NASH” also refers to liver inflammation anddamage caused by a buildup of fat in the liver. “NASH” also refers to asubtype of nonalcoholic fatty liver disease (“NAFLD). In someembodiments, “NASH” may be synonymous with “NAFLD.”

The term “obesity” as used herein refers to a disorder involving excessbody fat that increases the risk of health problems. The term “obesity”also refers to weight that is higher than what is considered a healthyweight for a given height. The term “obesity” also refers to a BMIgreater than 30.0. As used herein, body mass index (BMI) refers to aperson's weight in kilograms divided by the square of height in meters.

Certain abbreviations are defined as follows: “Aib” refers to2-aminoisobutyric acid; “AMY1” refers to amylin receptor 1; “cAMP”refers to cyclic adenosine monophosphate; “CT” refers to calcitonin;“DCM” refers to dichloromethane; “DIEA” refers to diisopropylethylamine;“DIO” refers to diet induced obese; “DMF” refers to dimethylformamide;“FBS” refers to fetal bovine serum; “GPCR” refers G-protein coupledreceptor(s); “HEPES” refers to4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; “HTRF” refers tohomogeneous time resolved fluorescence; “IBMX” refers to1-methyl-3-isobutylxanthine; “Mtt” refers to 4-methyltrityl; “PBMC”refers to peripheral blood mononuclear cell; “PyAOP” refers to(7-azabenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate; “RP-HPLC” refers to reverse phase high pressureliquid chromatography; and “TFA” refers to trifluoroacetic acid.

Example 1: Preparation and Purification of Compound I and Compound II

Compound I and Compound II are made according to the following steps.First, Compound I is synthesized using Fluorenylmethyloxycarbonyl(Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-channel multiplexpeptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.).

Polystyrene Rink Amide AM LL resin (Novabiochem, sub: 0.33 meq/g,100-200 mesh, Cat #855045) is used for the synthesis at 0.13 mmol scale.

Standard sidechain protecting groups are used. Fmoc-Lys(Mtt)-OH) is usedfor the lysine at position 11. Fmoc groups are removed prior to eachcoupling step (2×7 minutes) using 20% piperidine in DMF. All amino acidcouplings are performed for 30 minutes at 50° C. using an equal molarratio of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM) andOxyma (0.9 mM), at a 7.7-fold molar excess over the theoretical peptideloading. N-termini is acetylated with 5% acetic anhydride, 5% DIEA inDMF for 30 minutes. Below is a schematic of Compound I (SEQ ID NO:1)

Then, the resin is thoroughly washed with DCM for 6 times to removeresidual DMF The Mtt protecting group on the lysine at position 11 isselectively removed from the peptide resin using two treatments of 30%hexafluoroisopropanol (Oakwood Chemicals) in DCM (2×40-minutetreatment).

Synthesis of Compound II is made according to the following steps.Subsequent attachment of the fatty acid-linker moiety is accomplished bycoupling of 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid(Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid α-t-butyl ester(Fmoc-Glu-OtBu, Ark Pharm, Inc.), mono-OtBu-eicosanoic acid (WuXiAppTec, Shanghai, China). 3-Fold excess of reagents (AA:PyAOP:DIEA=1:1:1mol/mol) are used for each coupling that is 1-hour long.

After the synthesis is complete, the peptide resin is washed with DCM,and then thoroughly air-dried. The dry resin is treated with 10 mL ofcleavage cocktail (TFA:water:triisopropylsilane, 95:2.5:2.5 v/v) for 2hours at room temperature. The resin is filtered off, washed twice eachwith 2 mL of neat TFA, and the combined filtrates are treated with4-fold cold diethyl ether (−20° C.) to precipitate the crude peptide.The peptide/ether suspension is then centrifuged at 3500 rpm for 2 minto form a solid pellet, the supernatant is decanted, and the solidpellet is triturated with ether two additional times and dried in vacuo.The crude peptide is solubilized in 20% acetonitrile/20% acetic acid/60%water and purified by RP-HPLC on a Waters Xselect Peptide CSH C18 prepcolumn 130A 5 um 19×150 mm PN 186007021) with a linear gradient using100% acetonitrile and 0.1% TFA/water buffer system (20-40% acetonitrilein 60 min). The purity of peptide is assessed using analytical RP-HPLCand pooling criteria is >95%. The main pool purity of Compound I isfound to be >99.0%. Subsequent lyophilization of the final main productpool yields the lyophilized peptide TFA salt. The molecular weight isdetermined by LC/MS. Average MW=3447.8 Da. Expected mass: (M+3H):1150.3, found 1150.0.

The main pool purity of Compound II is found to be >98.0%. Subsequentlyophilization of the final main product pool yields the lyophilizedpeptide TFA salt. The molecular weight is determined by LC/MS. AverageMW=4191.8 Da. Expected mass: (M+3H): 1398.2, found 1398.2. Below is aschematic of Compound II (SEQ ID NO:2).

Similar processes to those described above and known to those of skillin the art may be used to synthesize the peptide backbone, conjugate thefatty acid-linker moiety, examine the purity, and confirm the molecularweight of the inventive compound described herein.

Example 2: In Vitro Functional Activity

The AMY1 and CT receptors are GPCRs that are functionally coupled to Gαsproteins. Stimulation of these receptors results in an increasedproduction of intracellular cAMP, which can be detected using standardin vitro technologies. Human AMY1 or CT receptors are stably expressedin human urinary bladder cells (UM-UC-3) cells under control of a pcDNA(CALCR) or pCMV piggybac (RAMP1) expression vector. AMY1 cells arecultured in MEM 1X (Corning) supplemented with 10% FBS, 1%antibiotic/antimycotic solution, 1 mM sodium pyruvate, 1X MEM NEAA, 1XGlutaMAX-I, 200 μg/mL hygromycin B, and 0.4 μg/mL puromycin. CT cellsare cultured in the same medium except that it lacks the puromycin.Cultured cells are grown to 70% confluency, and then incubated overnightwith fresh medium.

On the assay day, 10 μL of assay buffer (phenol red free MEM (Corning,cat #17-305-CV), 0.1% casein, 0.5 mM IBMX, 5 mM HEPES, pH 7.4) isdispensed into each well of white poly-D-lysine coated 384-well plates(Corning cat #354661). Peptides diluted in DMSO are added (200 nL/well)in a 1:3 dilution series using ECHO acoustic liquid handler (Beckman).Cultured cells are detached with TrypLE Express (Gibco), resuspended inassay buffer, and 10 μL containing 1200 cells/well (hCT) or 1500cells/well (hAMY1) are dispensed into each well. The plates areincubated at room temperature for 1 hour.

The amount of intracellular cAMP is quantitated using HTRF technology(Homogeneous Time Resolved Fluorescence; Cisbio) as per vendorinstructions. Briefly, 10 μL cAMP-d2 conjugate and 10 μLanti-cAMP-cryptate conjugate in lysis buffer are incubated with thetreated cells at room temperature for 60 min. The HTRF signal isimmediately detected using an Envision plate reader (Perkin-Elmer) tocalculate the ratio of fluorescence at 665 to 620 nm. The raw data areconverted to cAMP amount (pmole/well) using a cAMP standard curvegenerated for each experiment. Relative EC₅₀ values are calculated fromthe top-bottom range of the concentration response curve defined using 1nM salmon CT (Bachem) as the maximum and buffer alone as the minimumwith a four-parameter logistic curve fitting program (Genedata Screener®v12.0.4). The compounds of the present application show activity at theamylin and calcitonin receptors as shown in Table 1.

TABLE 1 Activity of Compounds I and II at Amylin and CalcitoninReceptors. Compound hAMY1 cAMP EC50 (pM) hCT cAMP EC50 (pM) Compound I49.3 ± 8.04 (n = 4)  31.7 ± 5.8 (n = 3) Compound II 49.4 ± 5.14 (n = 7)40.9 ± 14.7 (n = 4)  Pramlintide  44.0 ± 4.79 (n = 22)  2320 ± 408 (n =18) Human calcitonin  397 ± 63.5 (n = 7) 106 ± 20.5 (n = 6)

Example 3: In Vivo Efficacy in Normal Rat Models

Male Sprague Dawley (SD) rats from Envigo Laboratories weighingapproximately 300 grams are used to evaluate the effects of Compound IIon acute reductions in food intake and body weight loss in vivo during a96-hour evaluation study. The rats are maintained under approved AnimalCare and Use protocols for Lilly Research Laboratories and are housedindividually on a 12 hour reverse light cycle from 10 p.m. to 10 a.m.The morning of the study, baseline animal body and food weights aremeasured and vehicle (20 mM Tris pH8, 50 mg/mL D-mannitol, 0.02% PS80)or Compound II (SEQ ID NO:2) alone in vehicle (20 mM Tris pH8, 50 mg/mLD-mannitol, 0.02% PS80) are subcutaneously administered at differentdoses.

Animal body and food weights are measured at 24-, 48-, 72- and 96-hourspost-administration. The daily food intake and the percent decrease inbody weight are calculated. The results are listed below in Tables 2 and3. All data is indicated as the mean daily food intake and percent bodyweight decrease. As demonstrated by the results, Compound II has aneffect to reduce food consumption and reduce body weight acutely and ina dose-dependent manner. Also, even very low doses show efficacy inlowering food intake and body weight.

TABLE 2 Effect of Compound II on Daily Food Intake in Sprague-DawleyRats. Dose Daily Food Intake (grams) (nmol/kg) 0 h 24 h 48 h 72 h 96 hControl 0.00 18.58 20.42 19.88 20.12 0.1 0.00 16.12 19.65 19.93 21.600.3 0.00 15.55 18.65 20.47 21.32 1 0.00 13.72 16.92 19.77 21.03 3 0.005.32 10.22 14.88 18.78 10 0.00 2.77 0.87 9.62 15.45 30 0.00 3.75 0.305.67 14.85

TABLE 3 Effect of Compound II on % Change in Body Weight inSprague-Dawley Rats. Dose % Change in Body Weight (nmol/kg) 0 h 24 h 48h 72 h 96 h Control 0.00 −0.38 0.54 1.27 1.61 0.1 0.00 −0.89 −0.16 0.612.06 0.3 0.00 −0.91 −0.64 0.39 1.50 1 0.00 −1.82 −1.44 −0.38 0.57 3 0.00−3.63 −4.68 −4.66 −3.26 10 0.00 −5.67 −10.02 −9.88 −8.95 30 0.00 −6.24−11.60 −12.43 −11.74

Example 4: In Vivo Efficacy in Diet Induced Obese (DIO) Rats

This study is conducted to investigate the effect of Compound II fordiabetes and/or obesity conditions in DIO rats. Diet-induced obese (DIO)male Long Evans (Envigo) rats 24 to 30 weeks old, maintained on acalorie rich diet since arrival at Lilly (TD95217; Teklad, Madison,Wis.), are used in the following studies. Animals are individuallyhoused in a temperature-controlled (24° C.) facility with 12-hourlight/dark cycle (lights on 2200) and free access to food (TD95217) andwater.

The rats are randomized according to their body weight, so that eachexperimental group of animals would have similar body weight. The bodyweights range from 514 to 710 grams.

Each groups contains five rats. Vehicle and Compound II (0.1 and 100nmol/kg) dissolved in vehicle (Tris pH8, (50 mg/mL D-mannitol)+0.02%ps80) are administered by subcutaneous (SC) injection (1 mL/kg) to adlibitum fed DIO rats 30 to 60 minutes prior to the onset of the darkcycle every 3 days for 15 days. SC injections are made on Day 1, 4, 7,10 and 13. Body weight and food intake are measured daily throughout thestudy. Absolute changes in body weight are calculated by subtracting thebody weight of the same animal prior to the first injection of molecule.Body composition was assessed by quantitative nuclear magnetic resonance(QMNR, EchoMRI LLC, Houston, Tex.) on Days −1 (one day prior totreatment) and 15.

At the end of the study, blood is collected to measure blood glucose andplasma insulin. Blood glucose is measured by AccuChek glucometers(Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD,Rockville, Md.).

All data are presented as mean±SEM of 5 animals per group. Statisticalanalysis is performed using repeated measures ANOVA, followed byDunnett's method for multiple comparisons. Significant differences areidentified at p<0.05.

Compound II reduces body weight and food intake in male DIO rats in adose-dependent manner. Reduced body weight is likely primarily due toreduction in fat mass. In addition to substantial weight loss, reducedserum glucose and reduced insulin are observed with treatment usingCompound II in a dose-dependent manner as shown in Tables 4 and 5.

Body composition measurements are done on Day 0 and Day 14. The changefrom the initial measurement is presented as Measurement on Day0-Measurement on Day 14. All data are presented as mean±SEM of 5 animalsper group from Day 14. Statistical analysis is performed using repeatedmeasures ANOVA, followed by Dunnett's method for multiple comparisons.Significant differences are identified at p<0.05.

TABLE 4 The effect of Compound II on body weight, cumulative food intakeand fat mass in DIO rats on Day 15 Compound Body Weight Cumulative FoodFat Mass (dose) Change (g) Intake (g) Change (g) Vehicle  8.36 ± 3.69236.38 ± 6.08  3.58 ± 1.0 (1 mL/kg, SC) Compound II  4.32 ± 1.66 235.58± 6.34   4.93 ± 2.96 (0.1 nmol/kg) Compound II −18.74 ± 3.31* 197.14 ±14.35  −10.62 ± 2.80* (0.3 nmol/kg) Compound II −38.30 ± 5.67* 163.56 ±13.40* −22.42 ± 4.74* (1 nmol/kg) Compound II −59.84 ± 5.32* 125.70 ±12.36* −38.09 ± 3.64* (3 nmol/kg) Compound II −73.06 ± 4.27* 110.12 ±11.24* −43.26 ± 0.81* (10 nmol/kg) Compound II −77.24 ± 3.41* 103.14 ±13.53* −41.94 ± 6.05* (30 nmol/kg) Compound II −85.84 ± 3.78*  89.36 ±10.45* −40.77 ± 3.46* (100 nmol/kg) *Significant differences areidentified at p < 0.05

TABLE 5 The effect of Compound II treatment on fasting blood glucose,insulin, insulin resistance index (HOMA-IR = fasting glucose[mmol/L] ×Fasting Insulin [μU/ml])/22.5) and glucose area under the curve for 60minutes (AUC 60 min) during oral glucose tolerance test (OGTT) in DIOrats Fasting Fasting HOMA-IR Glucose (mg/dL) Compound Glucose Insulin[FI] [(FG × AUC (60 min) (dose) [FG](mmol/L) (μU/ml) FI)/22.5] duringOGTT Vehicle 7.10 ± 0.40 39.08 ± 4.77  12.52 ± 1.95  14582.25 ± 1299.63(1 ml/kg, SC) Compound II 6.94 ± 0.22 41.68 ± 5.13  12.98 ± 1.69 14638.50 ± 607.28  (0.1 nmol/kg) Compound II 6.50 ± 0.13 35.06 ± 4.89 10.22 ± 1.56  11367.75 ± 486.94* (0.3 nmol/kg) Compound II 6.94 ± 0.4318.38 ± 3.53* 5.82 ± 1.31* 11367.75 ± 703.77* (1 nmol/kg) Compound II6.78 ± 0.28 23.84 ± 2.15* 7.26 ± 0.93* 11189.25 ± 657.87* (3 nmol/kg)Compound II 7.04 ± 0.20 28.08 ± 2.51  8.72 ± 0.62  10959.75 ± 507.17*(10 nmol/kg) Compound II 6.02 ± 0.38 15.16 ± 3.63* 4.02 ± 0.91*  9936.00± 752.37* (30 nmol/kg) Compound II  5.86 ± 0.32* 11.92 ± 2.46* 3.20 ±0.76* 10014.00 ± 538.58* (100 nmol/kg) *Significant differences areidentified at p < 0.05

Example 5: Pharmacokinetic Behavior of Compound II

Plasma concentration of Compound II is determined by a qualified LiquidChromatography Mass Spectrometry (LC/MS) method at Q Squared SolutionsBioSciences LLC, Ithaca, N.Y. Compound II and an analog as an internalstandard are extracted from 100% species-specific plasma using proteinprecipitation followed by solid phase extraction. The intact mass ofCompound II which includes peptide plus acyl chain is detected by aQ-Exactive Orbitrap™ mass spectrometer.

In a monkey pharmacokinetics study, male and female Cynomolgus monkeysare administered a single subcutaneous dose of 20 nmol/kg (0.084 mg/kg)of Compound II in Tris-mannitol buffer (pH 8.0) at a volume of 0.2mL/kg. Blood is collected predose and at 1, 3, 6, 12, 24, 48, 72, 96,120, 144, 168, 240, 336, 408, 504 hours postdose for pharmacokineticcharacterization. The results are shown in Table 6.

TABLE 6 Individual and Mean Plasma Pharmacokinetic Parameters Followinga Single 20 nmol/kg Subcutaneous Dose of Compound II to Male and FemaleCynomolgus Monkeys Cmax AUC_(0-inf) CL/F Animal T_(1/2) Tmax (nmol/(hr*nmol/ (mL/hr/ Compound ID (hr) (hr) L) L) kg) Compound II P0001 18512 198 50355 0.40 P0101 170 72 138 44678 0.45 Mean 178 42 168 47516 0.42Abbreviations: T_(1/2) = half-life, T_(max) = time to maximumconcentration, C_(max) = maximum observed plasma concentration,AUC_(0-inf) = area under the curve from time 0 hours to infinity, CL/F =clearance/bioavailability.In a rat pharmacokinetic study, male Sprague Dawley rats areadministered a single subcutaneous dose of 20 nmol/kg (0.084 mg/kg) ofCompound II in Tris-mannitol buffer (pH 8.0) at a volume of 0.2 mL/kg.Blood is collected at 1-, 3-, 6-, 12-, 24-, 48-, 72-, 96-, 120-, and144-hours post-dose for pharmacokinetic characterization. The resultsare shown in Table 7.

TABLE 7 Individual and Mean Plasma Pharmacokinetic Parameters Followinga Single 20 nmol/kg Subcutaneous Dose of Compound II (SEQ ID NO: 2) toMale Sprague Dawley Rats Cmax AUC_(0-inf) CL/F Animal T_(1/2) Tmax(nmol/ (hr*nmol/ (mL/hr/ Compound ID (hr) (hr) L) L) kg) Compound IIR0001 47 48 88 8409 2.38 R0002 60 12 81 8702 2.30 R0003 67 12 99 86552.31 Mean 58 24 90 8588 2.33 SD 10 21 9 157 0.04 Abbreviations: T_(1/2)= half-life, T_(max) = time to maximum concentration, C_(max) = maximumobserved plasma concentration, AUC_(0-inf) = area under the curve fromtime 0 hours to infinity, CL/F = clearance/bioavailability.

The extended half-lives demonstrated by Compound II in CynomolgusMonkeys show therapies with once weekly dose of Compound II is possiblein patients.

Example 6: In Vivo Efficacy in Diet Induced Obese (DIO) Rats of CompoundII in Combination with Other Incretin Compounds

This study is conducted to investigate the effect of Compound II fordiabetes and/or obesity conditions in DIO rats when administered incombination with other incretin compounds, including a GLP-1 agonist(Compound III), oxyntomodulin analog (Compound IV), and a triagonist ofglucagon, GLP-1, and GIP (Compound V). Diet-induced obese (DIO) maleLong Evans (Envigo) rats, maintained on a calorie rich diet sincearrival at Lilly (TD95217; Teklad, Madison, Wis.), are used in thefollowing studies. Animals are individually housed in atemperature-controlled (24° C.) facility with 12-hour light/dark cycle(lights on 2200) and free access to food (TD95217) and water.

The rats are randomized according to their body weight, so that eachexperimental group of animals would have similar body weight. The bodyweights range from 529 to 823 grams.

Each group contains five rats. Vehicle and Compound II (1 nmol/kg) aredissolved in vehicle (40 mM Tris-HCl pH8+0.02% PS80) and areadministered by subcutaneous (SC) injection (1 mL/kg) to ad libitum fedDIO rats 30 to 90 minutes prior to the onset of the dark cycle every 3days for 14 days. SC injections are made on Days 1, 4, 7, 10 and 13.Body weight and food intake are measured daily throughout the study.Absolute changes in body weight are calculated by subtracting the bodyweight of the same animal prior to the first injection of molecule.

At the end of the study, blood is collected to measure blood glucose andplasma insulin. Blood glucose is measured by AccuChek glucometers(Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD,Rockville, Md.).

All data are presented as mean±SEM of 5 animals per group. Statisticalanalysis is performed using one-way ANOVA, followed by Tukey's multiplecomparison test to compare treatment groups to vehicle group or eachother. Significant differences are identified at p<0.05.

TABLE 8 The Effect of Compound II With and Without Combinations withCompound III, Compound IV, or Compound V on Body Weight and CumulativeFood Intake. Body Weight Cumulative Food Treatment* Change (g)** Intake(g)*** Vehicle −3.42 ± 6.10 212.08 ± 16.84 (10 mL/kg) Compound II −53.84± 2.85* 137.38 ± 4.28* (1 nmol/kg) Compound III −54.20 ± 6.52* 150.04 ±5.71* (10 nmol/kg) Compound IV −50.24 ± 6.36* 165.86 ± 7.45* (10nmol/kg) Compound V −43.28 ± 2.54*  157.50 ± 10.63* (3 nmol/kg) CompoundII +  −85.64 ± 5.08*#+   92.66 ± 11.52*#+ Compound III Compound II + −85.43 ± 8.66*#  113.58 ± 9.19*# Compound IV Compound II +    −89.70 ±11.63*#+  97.16 ± 6.45*# Compound V *Treatments were subcutaneouslyadministered every three days on Day 1, 4, 7, 10 and 13. **Body weightmeasurements were made daily. Body weight change is the difference fromDay −1 to Day 14 represented as grams. ***Cumulative food intake was thetotal food consumed throughout 14-day treatment period. Statisticalanalysis was done by one-way ANOVA followed by Tukey's. *p < 0.05compared to vehicle group; #p < 0.05 compared to either Compound III,Compound IV or Compound V group; +p < 0.05 compared to Compound II.

Example 7: An Analogue of Compound II in Combination with an Agonist ofGIP-GLP

This study is conducted to investigate the effect of an analog ofCompound II for diabetes and/or obesity conditions in DIO rats whenadministered with Compound VI (SEQ ID NO:9), a dual agonist of GIP andGLP-1. Diet-induced obese (DIO) male Long Evans (Envigo) rats,maintained on a calorie rich diet since arrival at Lilly (TD95217;Teklad, Madison, Wis.), are used in the following studies. Animals areindividually housed in a temperature-controlled (24° C.) facility with12 hour light/dark cycle (lights on 2200) and free access to food(TD95217) and water.

The rats are randomized according to their body weight, so that eachexperimental group of animals would have similar body weight. The bodyweights range from 549 to 683 grams.

Each group contains five rats. Vehicle and Compound II (1 nmol/kg)dissolved in vehicle (10 mM Tris-HCl pH7.5, (50 mg/mL D-mannitol)+0.02%PS80) are administered by subcutaneous (SC) injection (1 mL/kg) to adlibitum fed DIO rats 30 to 90 minutes prior to the onset of the darkcycle every 3 days for 14 days. SC injections are made on Days 1, 4, 7,10 and 13. Body weight and food intake are measured daily throughout thestudy. Absolute changes in body weight are calculated by subtracting thebody weight of the same animal prior to the first injection of molecule.

At the end of the study, blood is collected to measure blood glucose andplasma insulin. Blood glucose is measured by AccuChek glucometers(Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD,Rockville, Md.).

All data are presented as mean±SEM of 5 animals per group. Statisticalanalysis is performed using one-way ANOVA, followed by Tukey's multiplecomparison test to compare treatment groups to vehicle group or eachother. Significant differences are identified at p<0.05.

Body Weight Cumulative Food Treatment* Change (g)** Intake (g)***Vehicle  6.18 ± 1.37 231.58 ± 1032  (10 ml/kg) Analog of Compound II−24.82 ± 3.24* 190.74 ± 6.56  (0.1 nmol/kg) Compound VI −18.10 ± 6.67*196.54 ± 16.98 (3 nmol/kg) Analog of Compound II +  −39.84 ± 2.20*#  174 ± 6.48* Compound VI *Treatments were subcutaneously administeredevery three days on Day 1, 4, 7, 10 and 13. **Body weight measurementswere made daily. Body weight change is the difference from Day −1 to Day16 represented as grams. ***Cumulative food intake was the total foodconsumed throughout 16-day treatment period. Statistical analysis wasdone by one-way ANOVA followed by Tukey's. *p < 0.05 compared to vehiclegroup; #p < 0.05 compared to GIP-706.An analogue of Compound II in combination with Compound VI caused moreweight loss than individual treatment which is likely mainlyattributable to significant decrease in cumulative food intake inducedby combination treatment.

Example 8: Immunogenicity Risk Assessment MAPPs Assay (MHC-AssociatedPeptide Proteomics):

Primary human dendritic cells from ten normal human donors are preparedfrom buffy coats by isolation of CD14+ cells and differentiated intoimmature dendritic cells by incubation with 20 ng/mL IL-4 and 40 ng/mLGM-CSF in complete RPMI media containing 5% Serum Replacement (ThermoFisher Scientific, cat #A2596101) for three days at 37° C. and 5% CO₂ asdescribed (Knierman et al., “The Human Leukocyte Antigen Class IIImmunopeptidome of the SARS-CoV-2 Spike Glycoprotein”, Cell Reports, 33,108454 (2020)). Three micromolar of test antibody is added toapproximately 5×10⁶ cells on day 4 and fresh media containing 5 μg/mL ofLPS to transform the cells into mature dendritic cells is exchangedafter 5-hour incubation. The matured cells are lysed in 1 mL of RIPAbuffer with protease inhibitors and DNAse the following day. The lysatesare stored at −80° C. until sample analysis.

An automated liquid handling system is used to isolate the HLA-IImolecules from thawed lysate using biotinylated anti-pan HLA class IIantibody (clone Tu39). The bound receptor-peptide complex is eluted with5% acetic acid, 0.1% TFA. The eluted MHC-II peptides are passed over aprewashed 10 k MWCO filter to remove high molecular weight proteins. Theisolated MHC-II peptides are analyzed by nano LC/MS using a Thermo easy1200 nLC-HPLC system with a Thermo LUMOS mass spectrometer. Theseparation used a 75 μm×7 cm YMC-ODS C18 column for 65-minute gradientwith a 250 nL/min flow rate and 0.1% formic acid in water as A solventand 80% acetonitrile with 0.1% formic acid as B solvent. Massspectrometry is run in full scan mode with 240,000 resolution followedby a 3 second data dependent MS/MS cycle comprised of ion trap rapidscans with HCD and EThcD fragmentation.

Peptide identifications are generated by an internal proteomics pipeline(Higgs et al., “Label-free LC-MS method for the identification ofbiomarkers”, Methods in Molecular Biology, 428, 209-230 (2008)) usingmultiple search algorithms with no enzyme search parameter against abovine/human database containing the test antibody sequences. A KNIMEworkflow is used to process the identification files for the samples.Peptides identified from the test articles are aligned against theparent sequence. A summary is created for all donors that annotates thepercent of donors that display non-germline residues, the number ofdifferent regions that display peptides with non-germline residues andthe depth of peptide display at each region with non-germline residues.Increases in the extent of display of non-germline peptides isassociated with increased risk for immunogenicity. Results for CompoundII are shown in Table 9 and suggest a low level of immunogenicity riskassociated with Compound II.

TABLE 9 MAPPs Results Total # of non- Total # of non- Test % # ofgermline residues germline peptides Compound Donors clusters from allclusters from all clusters Compound II 0% 0 n/a* n/a* *n/a designatestotals are not applicable because there were no donors displayingnon-germline residues.

T Cell Proliferation Assay

This assay assesses the ability of test candidate to activate CD4+ Tcells by inducing cellular proliferation as described (Walsh et al.,“Post-hoc assessment of the immunogenicity of three antibodies revealsdistinct immune stimulatory mechanisms”, mAbs, 12, 1764829 (2020)).Cryopreserved PBMCs were used from ten healthy donors and the CD8+ Tcells were depleted from the PBMCs and labeled with 1 μMCarboxyfluorescein Diacetate Succinimidyl Ester (CFSE). PBMCs wereseeded at 4×10⁶ cells/ml/well in AIM-V media (Life Technologies, cat#12055-083) containing 5% CTS™ Immune Cell SR (Gibco, cat #A2596101) andtested in triplicate in 2.0 mL containing the different test articles,media control, keyhole limpet haemocyanin (KLH; positive control),lixisenatide (assay positive control), or Compound II. Cells werecultured and incubated for 7 days at 37° C. with 5% CO₂. On day 7,samples were stained with the following cell surface markers: anti-CD3,anti-CD4, anti-CD14, anti-CD19, and DAPI for viability detection by flowcytometry using a BD LSRFortessa™, equipped with a High ThroughputSampler (HTS). Data was analyzed using FlowJo® Software (FlowJo, LLC,TreeStar) and a Cellular Division Index (CDI) was calculated. Briefly,the CDI for each test candidate's MAPPs-derived peptide cluster wascalculated by dividing the percent of proliferating CFSE^(dim)CD4+ Tcells from peptide-stimulated wells by the percent of proliferatingCFSE^(dim)CD4+ T cells in the unstimulated wells. A CDI of ≥2.5 wasconsidered to represent a positive response. A percent donor frequencyacross all donors was evaluated. All donors produced a positive T cellresponse against KLH (100%). The clinical immunogenic positive controllixisenatide induced a positive T cell response of 50% of the cohort inthis study. This falls within the expected range for this assay (40-90%Positive Donor Frequency). Results for Compound II are shown in Table 10and suggest a low level of immunogenicity risk for Compound II.

TABLE 10 The Frequency of CD4+ T cell Responses Median Median CDI CDIMolecule % Positive (Positive (All Range Number of Tested Donors Donors)donors) High Low donors KLH 100.0 36.2 36.2 171.4 4.5 8/8 Lixisenatide50 3.0 2.5 5.7 1.2 4/8 Compound II 12.5 19.7 1.0 19.7 0.7 1/8

Sequences Compound 1 (SEQ ID NO: 1)Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH₂Compound II (SEQ ID NO: 2)Acetyl-ASHLSTAVLGK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO-(CH₂)₁₈-CO₂H)LS-Aib-ELHKLEDYPRTDVGAESP-NH₂ Pramlintide (SEQ ID NO: 3)KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH₂with a disulfide bond between Cys 2 and Cys 7Human calcitonin (SEQ ID NO: 4) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH₂with a disulfide bond between Cys 1 and Cys 7Human amylin (SEQ ID NO: 5) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH₂with a disulfide bond between Cys 2 and Cys 7Compound III (SEQ ID NO: 6)H-Aib-EGTFTSDVSSYLEGQAAK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γGlu)-CO-(CH₂)₁₆-CO₂H)EFIAWLVRGRG Compound IV (SEQ ID NO: 7)H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γGlu)-CO-(CH₂)₁₈-CO₂H)EFVEWLLEGGPSSG-NH₂ Compound V (SEQ ID NO: 8)Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH₂)₁₈-CO₂H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH₂Compound VI (SEQ ID NO: 9)Y-Aib-EGTFTSDYSI-Aib-LDKIAQK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γGlu)₂-CO-(CH₂)₁₈-CO₂H)A-(1Nal)-VQWLIAGGPSSGAPPPS-NH₂Compound VII (SEQ ID NO: 10) YX₁EGTFTSDYSIX₂LDKIAQKAFVQWLIAGGPSSGAPPPSwherein X₁ is Aib; X₂ is Aib; K at position 20 is chemically modified throughconjugation to the epsilon-amino group of the K side-chain with (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γGlu)₁-CO-(CH₂)₁₈-CO₂H; and the C-terminal amino acid is amidatedas a C-terminal primary amide. CAS Registry Number: 2023788-19-2Lixisenatide (SEQ ID NO: 11)HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH₂CAS Registry Number: 320367-13-3 Compound VIII (SEQ ID NO: 12)HAEGTFTSDVSSYLEGQAAK(γGlu-CO-(CH₂)₁₄-CH₃)EFIAWLVRGRGCAS Registry Number: 204656-20-2 Compound IX (SEQ ID NO: 13)Dulaglutide is a human GLP-1 receptor agonist which comprises a dimer of aGLP-1 analog fused at its C-terminus via a peptide linker to the N-terminus of an analogof an Fc portion of an immunoglobulin, and is identified by CAS registry number923950-08-7, which provides the following chemical name: 7-37-Glucagon-like peptide I[8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) fusion protein with peptide(synthetic 16-amino acid linker) fusion protein with immunoglobulin G4 (synthetichuman Fc fragment), dimer. Each monomer of dulaglutide has the amino acid sequenceset forth in SEQ ID NO: 13:HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG

We claim: 1-13. (canceled)
 14. A method of treating a condition selectedfrom the group consisting of diabetes, obesity, NASH, and dyslipidemia,in a patient in need thereof, comprising administering to the patient aneffective amount of a compound comprising SEQ ID NO: 1, or apharmaceutically acceptable salt thereof, in combination with aneffective amount of a GLP-1 agonist.
 15. The method of claim 14, whereinthe GLP-1 agonist is selected from the group consisting of Compound III(SEQ ID NO:6), Compound VIII (SEQ ID NO:12), and Compound IX (SEQ IDNO:13).
 16. The method of claim 14, wherein the compound comprising SEQID NO:1 is administered in separate, simultaneous, or sequentialcombination with the GLP-1 agonist.
 17. The method of claim 15, whereinthe compound comprising SEQ ID NO:1 is administered in separate,simultaneous, or sequential combination with the GLP-1 agonist.
 18. Amethod of treating a condition selected from the group consisting ofdiabetes, obesity, NASH, and dyslipidemia, in a patient in need thereof,comprising administering to the patient an effective amount of acompound comprising SEQ ID NO: 1, or a pharmaceutically acceptable saltthereof, in combination with an effective amount of a dual GIP/GLP-1agonist.
 19. The method of claim 18, wherein the dual GIP/GLP-1 agonistis selected from the group consisting of Compound VI (SEQ ID NO:9) andCompound VII (SEQ ID NO: 10).
 20. The method of claim 18, wherein thecompound comprising SEQ ID NO:1 is administered in separate,simultaneous, or sequential combination with the dual GIP/GLP-1 agonist.21. The method of claim 19, wherein the compound comprising SEQ ID NO:1is administered in separate, simultaneous, or sequential combinationwith the dual GIP/GLP-1 agonist.
 22. A method of treating a conditionselected from the group consisting of diabetes, obesity, NASH, anddyslipidemia, in a patient in need thereof, comprising administering tothe patient an effective amount of a compound comprising SEQ ID NO:1, ora pharmaceutically acceptable salt thereof, in combination with aneffective amount of a tri-agonist of glucagon, GIP, and GLP-1.
 23. Themethod of claim 22, wherein the tri-agonist of glucagon, GIP, and GLP-1is Compound V (SEQ ID NO:8).
 24. The method of claim 22, wherein thecompound comprising SEQ ID NO:1 is administered in separate,simultaneous, or sequential combination with the tri-agonist ofglucagon, GIP, and GLP-1.
 25. The method of claim 23, wherein thecompound comprising SEQ ID NO:1 is administered in separate,simultaneous, or sequential combination with the tri-agonist ofglucagon, GIP, and GLP-1.
 26. A method of treating a condition selectedfrom the group consisting of diabetes, obesity, NASH, and dyslipidemia,in a patient in need thereof, comprising administering to the patient aneffective amount of a compound comprising SEQ ID NO:1, or apharmaceutically acceptable salt thereof, in combination with aneffective amount of an analog of oxyntomodulin.
 27. The method of claim26, wherein the analog of oxyntomodulin is Compound IV (SEQ ID NO:7).28. The method of claim 26, wherein the compound comprising SEQ ID NO:1is administered in separate, simultaneous, or sequential combinationwith the analog of oxyntomodulin.
 29. The method of claim 27, whereinthe compound comprising SEQ ID NO:1 is administered in separate,simultaneous, or sequential combination with the analog ofoxyntomodulin.